Method of screening a plurality of single secreting cells for functional activity

ABSTRACT

This invention generally relates to methods, devices and kits for screening a plurality of single secreting cells for functional activity of the secreted molecules by measuring the amount of reporter gene mRNA produced in one or more reporter cells in response to the secreted molecules.

STATEMENT OF RELATED CASES

The present application is a continuation of U.S. application Ser. No.14/364,520, filed Jun. 11, 2014, which is a national phase filing under35 U.S.C. 371 of International Patent Application No. PCT/US12/69205,filed Dec. 12, 2012, which claims the benefit of priority to U.S.Provisional Application No. 61/630,493, filed Dec. 13, 2011.

FIELD OF THE INVENTION

This invention generally relates to methods, devices and kits forscreening a plurality of single secreting cells for functional activityof secreted molecules.

BACKGROUND

Cells that secrete molecules are an important source of therapeuticallyimportant biological molecules, and thus it is often valuable to screensecreted molecules from a population of secreting cells for certainfunctional properties. Useful secreting cells may be, for example,murine or human plasma cells or other antibody secreting cells, wherethe secreted antibody may block the binding of a cytokine to its cognatereceptor thereby disrupting a signal transduction event that mayunderlie the physiopathology of a disease. Therefore, such blockingantibodies possess tremendous therapeutic value and hence are ofinterest to the pharmaceutical industry. Alternatively, engineeredbioactive molecules such as a cytokine with a variant amino acid at astrategic residue can be screened for enhanced binding to a cognatereceptor. Currently, such functional screening is a laborious proceduretypically done with a large number of cells in 96-well plates.

Much work has been done to engineer cells to “report” when a ligandbinds to a receptor expressed on the surface of the cells. Engineeringhas been desirable because the cells may not have readily assayablechanges in response to such a binding event. Therefore, certain genesare chosen as reporters because the characteristics they confer whenexpressed are easily identified and measured. Some reporter cells arecomprised of reporter genes encoding readily assayable proteins underthe control of well-characterized transcriptional regulatory elementsresponding as part of a well-known signal transduction pathway. Examplesof common reporter genes are LacZ (encoding beta-galactosidase), luc(encoding firefly luciferase), luxCDABE (encoding bacterial luciferase)and GFP (encoding jellyfish green fluorescent protein) (Ghim et al., BMBreports, 2010, 451:460). One of the most commonly used signaltransduction pathways is the NF-κB activation pathway. Reporter genesare typically placed under the control of NF-κB regulatory elements andare used to screen molecules interacting with the relevant receptor,which causes activation of NF-κB and is manifested by expression of thereporter genes.

There are many limitations to each of the aforementioned reporter genes.The lacZ assay requires expensive and potentially toxic chemicals, theluc assay requires expensive luciferin, bacterial luciferase cannot beused in eukaryotic cells, and GFP is so stable it cannot be used toreport short term negative processes where the GFP signal needs todiminish quickly (Ghim et al., supra). Since the above-stated reportersystems typically are used on a large population of cells, it isdifficult to employ them in nanofabricated constructs on a limitednumber of cells. Furthermore, these reporter assays require translationof the protein products to visualize the output, which often takes hoursor even up to a day.

In addition to the above-mentioned reporter systems utilizing expressedproteins, microarrays have been used to investigate multi-gene mRNAexpression between multiple populations of tissues or cells. Microarraysallow many mRNA sequences to be sampled at the same time, but because ofthe expense of preparation and handling the number of tissue samples isgenerally small (less than 20) and the cell population large (more thana million cells). Therefore, the use of mRNA expression as a reportersystem is a serial and labor intensive process.

In order to correct the deficiencies of the current state of the art, amethod is needed where a single cell secreting a biologically activemoiety may be tested by one or more reporter cells and/or one or moretypes of reporter cells, and where an artificial reporter gene(s) doesnot need to be engineered into the reporter cell(s) to read out theresponse upon a binding event between a receptor and the active moiety,or upon disruption of binding. The present invention generally relatesto methods, devices and kits directed to these and similar utilities,such methods, devices and kits being further described herein.

SUMMARY OF THE INVENTION

This invention generally relates to methods, devices and kits forscreening a plurality of single secreting cells for functional activityof molecules secreted by them by measuring the Normalized Copy Number(NCN) of mRNA from one or more reporter genes produced by one or moretypes of reporter cells in response to the secreted molecules acting asa receptor agonist, as a receptor antagonist, or as a modulator. In oneembodiment, a plurality of secreting cells are spread and settled into aplurality of microwells with a majority of microwells containing asingle secreting cell. One or more types of reporter cells are alsoplaced in the microwells. Molecules secreted by the secreting cells arecaptured on one or more support surfaces brought into contact with themicrowells, and at the same time the secreted molecules are allowed tointeract with the reporter cells in the microwells. The secretedmolecules may act as a receptor agonist or receptor antagonist, andsubsequently the secreting cells and reporter cells are lysed releasingmRNA encoding the secreted molecules, the mRNA from one or more reportergenes in one or more types of reporter cells, and housekeeping gene(s)from the reporter cells. The mRNAs are captured on an oligonucleotidecapture support containing mRNA capture oligos comprising uniquenucleotide tags and, optionally, a random code at each location on theoligonucleotide capture support corresponding to each microwelllocation. The captured mRNA on the oligonucleotide capture support isconverted into cDNA incorporating the tags and optionally the randomcode, and the tagged and coded cDNA is sequenced using NGS technology.In addition, the binding kinetic properties of the secreted molecules toone or more specific moieties are measured.

The mRNA sequence encoding the secreted molecules is then associatedwith the measured binding kinetic properties and the Absolute CopyNumber (ACN) of the mRNA from one or more reporter genes of the reportercells is determined. Furthermore, the ACN of the mRNA of housekeepinggene(s) of the reporter cells is determined and used to calculate theNCN of mRNA of one or more reporter genes of the reporter cells. If morethan one type of reporter cells is used, typically the ACN of one ormore housekeeping genes is determined for each type of reporter cell.The NCN of the mRNA of one or more reporter genes of the reporter cellsassociated with a given secreted molecule in a microwell can be used toassess the secreted molecule's functional activity when compared to theNCN of the mRNA of one or more reporter genes of the reporter cellsassociated with other secreted molecules in a microwell or the NCN ofthe mRNA of one or more reporter genes of the reporter cells associatedwith an empty microwell.

In another embodiment, a plurality of secreting cells are spread andsettled into a plurality of microwells with a majority of microwellscontaining a single secreting cell. One or more types of reporter cellsare also placed in the microwells. Molecules secreted by the secretingcells are captured on one or more support surfaces brought into contactwith the microwells, at the same time the secreted molecules are allowedto interact with the reporter cells in the microwells as a modulator inthe presence of a naturally-occurring receptor agonist or receptorantagonist. Subsequently the secreting cells and reporter cells arelysed releasing their mRNA. The mRNA encoding the secreted molecules,the mRNA from one or more reporter genes in one or more types ofreporter cells, and housekeeping gene(s) from each type of the reportercells are captured on an oligonucleotide capture support containing mRNAcapture oligos comprising unique nucleotide tags and random code at eachlocation on the oligonucleotide capture support corresponding to eachmicrowell location. The captured mRNA on the oligonucleotide capturesupport is converted into cDNA incorporating the tags and optionallyrandom code, and tagged and coded cDNA is sequenced using NGStechnology. In addition, the kinetic properties of the secretedmolecules to one or more specific moieties are measured. The mRNAsequence encoding the secreted molecules is associated with the measuredkinetic properties and Absolute Copy Number (ACN) of the mRNA of one ormore reporter genes from each type of reporter cells is determined. TheACN of the mRNA of housekeeping gene(s) of each type of reporter cellsis determined and is used to calculate the NCN of mRNA of one or morereporter genes from each type of reporter cells. The NCN of the mRNA ofone or more reporter genes of the reporter cells associated with a givensecreted molecule in a microwell can be used to assess the secretedmolecule's functional activity when compared to the NCN of the mRNA ofone or more reporter genes of the reporter cells associated with othersecreted molecules in a microwell or the NCN of the mRNA of one or morereporter genes of the reporter cells associated with an empty microwell.

Thus, the present invention provides an efficient method for analyzing alarge number of secreting cells individually in a parallel manner ratherthan analyzing in a serial fashion a cell population and reporting theaverage measurement for the population. Each microwell, due to its smalldimensions in the range of microns, facilitates rapid reaction rates,such as mRNA hybridization or capture of proteins. The proteins capturedin the present invention are captured in a way to form an addressablearray on a solid surface where the kinetic properties of the capturedproteins can be analyzed en masse for binding affinity when reacted withlabeled affinity ligands. The mRNA subsequently captured on anoligonucleotide capture support is from two sources: a) the secretingcell, for example, mRNA encoding the light and heavy chains of amonoclonal antibody secreted by a plasma cell, and b) one or more typesof reporter cell(s). Although mRNA from an engineered reporter geneencoding a readily-assayable protein may be captured, reporter cellscontaining engineered reporter genes are not necessary in the presentinvention. This enables the use of un-engineered cell lines or evencells from a primary culture as reporter cells. The mRNA from one ormore reporter genes (either engineered reporter genes or endogenousreporter genes) capable of responding to a receptor agonist, a receptorantagonist, or a modulator, is captured and the NCN of the mRNA can bequantified by NGS and effectively used in analyzing the therapeuticutility of the secreted molecules.

Thus, in some embodiments, the present invention provides a method ofmeasuring the functional activity of a secreted molecule secreted by asecreting cell consisting of: placing one or more reporter cells intomicrowells; placing a plurality of secreting cells into the microwellssuch that a single secreting cell occupies a single microwell; allowingthe secreting moieties to interact with the reporter cells; capturingand measuring selected mRNA from the reporter cells; and comparing thecaptured and measured selected mRNA to housekeeping mRNA within thereporter cells and thereby determining if the secreted moieties modulatethe production of the selected mRNA within the reporter cells.

Other embodiments of the present invention provide a method of measuringa response of a reporter cell to a binding of a ligand to a receptorcomprising the steps of:

depositing into microwells a plurality of reporter cells, the reportercells express a receptor on their surface; depositing into themicrowells a plurality of secreting cells such that, on average, asingle secreting cell occupies a single microwell; allowing thesecreting cells to secrete a secreted molecule; optionally introducinginto the microwells a ligand that interacts with the receptor; lysingthe reporter cells; capturing mRNA of one or more reporter genes andoptionally mRNA of one or more housekeeping genes from the reportercells onto an oligonucleotide array placed in close proximity with thetop of the microwells, the oligonucleotide array containing one or moreunique DNA tags and an optional random code; converting mRNA from thereporter cells to cDNA, the mRNA coding for genes responding to theligand-receptor binding and optionally for housekeeping genes notresponding to ligand-protein binding; sequencing the cDNA and using theDNA tags and optional random codes to measure the response of thereporter cell to the binding of the ligand to the receptor.

In some aspects of these methods, the secreted molecule is an antibody.In some aspects, the receptor is a membrane bound protein permanentlybound to the lipid bilayer, a peripheral membrane protein temporarilyassociated with lipid bilayer or an integral membrane protein, or alipid-anchored protein bound to lipid bilayer bound through lipidatedamino acid residues.

In some aspects, the volume of said microwells is between 10 and 1000picoliters, and in some aspects, the number of reporter cells depositedinto said microwells is between 1 and 500 cells per microwell. In someaspects, the number of nucleotides in said DNA tag is more than 6nucleotides, and in some aspects, the number of nucleotides in saidrandom code is more than 4 nucleotides. Further aspects of theembodiments provide DNA tags with unique optional random codes used tocompute the ACN of the reporter gene and the ACN of the housekeepinggene, and in some aspects, the NCN of the ligand receptor binding iscomputed from the ration of the ACN of the reporter gene to the ACN ofthe housekeeping gene.

Other embodiments of the present invention provide a system or devicefor measuring a response of a reporter cell to a binding of a ligand toa receptor comprising the steps of: depositing into microwells aplurality of reporter cells, the reporter cells express a receptor ontheir surface; depositing into the microwells a plurality of secretingcells such that, on average, a single secreting cell occupies a singlemicrowell; allowing the secreting cells to secrete a secreted molecule;optionally introducing into the microwells a ligand that interacts withthe receptor; lysing the reporter cells; capturing mRNA of one or morereporter genes and optionally mRNA of one or more housekeeping genesfrom the reporter cells onto an oligonucleotide array placed in closeproximity with the top of the microwells, the oligonucleotide arraycontaining one or more unique DNA tags and an optional random code;converting mRNA from the reporter cells to cDNA, the mRNA coding forgenes responding to the ligand-receptor binding and optionally forhousekeeping genes not responding to ligand-protein binding; andsequencing the cDNA and using the DNA tags and optional random codes tomeasure the response of the reporter cell to the binding of the ligandto the receptor.

Yet another embodiment of the invention provides a method of measuringthe functional activity of a secreted molecule secreted by a secretingcell consisting of: placing a plurality of secreting cells intomicrowells such that a single secreting cell occupies a single microwelland is secreting molecules; placing one or more reporter cells of one ormore types into microwells; allowing copies of the secreted molecules tointeract with a receptor of the reporter cells; capturing mRNA from oneor more of the reporter genes of the reporter cells and capturing mRNAfrom one or more of the housekeeping genes of the reporter cells with anoligonucleotide capture support containing mRNA capture oligos on eachfeature comprising a unique DNA tag; converting captured mRNA into cDNAincorporating the DNA tag; sequencing the tagged and optionally codedcDNA by NGS; examining the sequenced cDNA from the one or more reportergenes and comparing it to the sequenced cDNA from the one or morehousekeeping genes of the reporter cells to determine the functionalactivity of a secreted molecule secreted by a secreting cell.

Yet an additional embodiment of the invention provides a method ofmeasuring a response of a reporter cell to a binding of a ligand to areceptor comprising the steps of: placing a plurality of ligands intomicrowells; placing one or more reporter cells of one or more types intothe microwells; allowing a ligand to interact with a receptor of thereporter cells; capturing mRNA from one or more of reporter genes of thereporter cells with an oligonucleotide capture support containing mRNAcapture oligos on each feature comprising a unique DNA tag; convertingcaptured mRNA into cDNA incorporating the DNA tag; sequencing the taggedcDNA by NGS; examining the one or more sequenced cDNA from reportergenes to determine the response of a reporter cell to the binding of aligand to a receptor.

Yet other embodiments of the present invention provide methods ofdetermining whether two similar sequences are original molecules in apopulation of nucleic acid molecules or are duplicates created duringsample preparation, e.g., PCR amplification, comprising: constructing apopulation of oligonucleotides containing a number of random codes,wherein the number of random codes is greater than the estimated numberof molecules in the population of nucleic acid molecules; incorporatinga single random code into every single molecule from a population ofnucleic acid molecules, amplifying the population of nucleic acidmolecules with the incorporated random codes; sequencing the amplifiedpopulation of nucleic acid molecules with the incorporated random codesusing a sequencing methodology that reports the sequence of individualnucleic acid molecules; and aggregating the reported sequences with thesame random code into sequences representing original sequences in saidpopulation of nucleic acid molecules. In some aspects of thisembodiment, the sequencing methodology is single molecule countingmethodology, and in preferred aspects, the sequencing methodology ishigh throughput sequencing techniques known in the art.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the methods and embodiments of the invention as more fullydescribed below.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures:

FIG. 1—Anatomy of two adjacent features and their mRNA capture probes ona DNA microarray

FIG. 2—Detection of a receptor agonist.

FIG. 3—Detection of an inverse agonist, a type of receptor agonist.

FIG. 4—Detection of a modulator.

FIG. 5—Detection of a receptor antagonist.

DEFINITIONS

“Absolute Copy Number (ACN)”—The copy number of the responding or steadystate levels of mRNA estimated by the count of independent cDNAmolecules enumerated by NGS sequences derived from one or more cellsbased on random codes.

“Normalized Copy Number (NCN)”—the ratio of the Absolute Copy Number ofthe reporter gene mRNA to the Absolute Copy Number of a singlehousekeeping gene or any gene selected for use as a benchmark, or theaverage Absolute Copy Number of two or more housekeeping genes or anygenes selected for use as a benchmark representing reporter cellhousekeeping activity. The reporter cell housekeeping activity can berepresented by the mRNA Absolute Copy Number of a single gene expressedby all reporter cells. Alternatively, the reporter cell housekeepingactivity can be represented by the average mRNA Absolute Copy Number oftwo or more housekeeping genes. In the latter case, an average valuemust be computed from the Absolute Copy Numbers of the two or morehousekeeping genes. The average value may be an arithmetic mean of theAbsolute Copy Numbers, or it may be a weighted mean, the weights derivedfrom the measured variation in Absolute Copy Numbers derived in previousruns, in the same run, or from published literature. Several weightingmethods are known to those skilled in the art (Press, Numerical Recipesin C. The Art of Scientific Computing, 2nd Edition 1992).

As used herein, “molecules” or “biomolecules” are broad representationsof moieties where not all of the interactions between atoms arecovalently bonded. For example, two strands of DNA held together byvarious forces including Watson-Crick hydrogen bonding and base stackingbalanced by the electrostatic repulsion of two phosphates either alongthe same strand or the opposite strands, are called a molecule as incDNA molecules. Molecules can also be used to refer to protein complexesheld together by various non-covalent bonding as in a complex formed byinteracting ligand and its cognate receptor. Molecules and moieties aresometimes used interchangeably.

As used herein, “secreted molecules” are molecular entities secreted bya secreting cell as defined below.

As used herein, a “secreting cell” is a cell that releases one or moresecreted molecules at rates that modify the local concentration of thesecreted molecules inside a microwell.

As used herein, “nanofabrication” is the design and manufacture ofdevices with dimensions measured in nanometers or micrometers. Anexample of nanofabrication is the construction of microwells in aflexible material. In one specific embodiment, SU-8 photoresist(MicroChem Corporation, Newton, Mass.) is spin-coated onto a 10 cmsilicon wafer in accordance with the manufacturer's recommendations. Apattern of 50 micron cubic micro-wells is exposed using ultravioletlight from a mask aligner (SÜSS MicroTec AG, Schleissheimer Str. 90,85748 Garching, Germany) in accordance with the manufacturer'srecommendations and developed using SU-8 developer (MicroChem).Polydimethylsiloxane (PDMS) (SylGard Elastomer, Ellsworth Adhesives,Germantown, Wis.) is mixed, cured and removed in accordance with themanufacturer's recommendations.

As used herein, a “biologically active agent” is any agent thatpossesses activity in a biological system. Examples of biologicallyactive agents include small molecule compounds; polypeptides, e.g.,proteins; siRNAs; and oligonucleotides. A plurality of such biologicallyactive agents would include, for example, 2 or more of such agents; insome cases, 3 or more agents; 5 or more agents; 10 or more agents; 20 ormore agents; 50 or more agents; 100 or more agents; 500 or more agents;1000 or more agents; 5000 or more agents; 10,000 or more agents; 30,000or more agents; 100,000 or more agents; or 1,000,000 or more of suchagents.

As used herein, the term “tag(s)” refers to a moiety that identifies thephysical location of its origin. In one preferred embodiment, a tag isan oligonucleotide tag(s) associated with a physical location on anoligonucleotide capture support that correlates to the position of amicrowell. The cDNA molecules having tags incorporated are hereinreferred to as “tagged cDNA” or “tagged cDNA molecules”.

The term “oligonucleotide” as used herein includes linear oligomers ofnatural or modified monomers or linkages, includingdeoxyribonucleosides, ribonucleosides, anomeric forms thereof, peptidenucleic acids (PNAs), and the like, capable of specifically binding to atarget polynucleotide by way of a regular pattern of monomer-to-monomerinteractions, such as Watson-Crick type of base pairing, base stacking,Hoogsteen or reverse Hoogsteen types of base pairing, or the like.Usually monomers are linked by phosphodiester bonds or analogs thereofto form oligonucleotides ranging in size from a few monomeric units,e.g. 3-4, to several tens of monomeric units, e.g. 40-60. Whenever anoligonucleotide is represented by a sequence of letters, such as“ATGCCTG,” it will be understood that the nucleotides are in 5′→3′ orderfrom left to right and that “A” denotes deoxyadenosine, “C” denotesdeoxycytidine, “G” denotes deoxyguanosine, and “T” denotes thymidine,unless otherwise noted. Usually oligonucleotides of the inventioncomprise the four natural nucleotides; however, they may also comprisenon-natural nucleotide analogs. It is clear to those skilled in the artwhen oligonucleotides having natural or non-natural nucleotides may beemployed, e.g. where processing by enzymes is called for, usuallyoligonucleotides consisting of natural nucleotides are required.

As used herein, a “selectable marker” is a gene introduced into a cell,especially a bacterium or to cells in culture that confers a traitsuitable for artificial selection. Selectable markers may be a type ofreporter gene used in laboratory microbiology, molecular biology, andgenetic engineering to change the phenotype of a cell so as to indicatethe success of a transfection or other procedure meant to introduceforeign DNA into a cell. Selectable markers may be antibiotic resistancegenes, genes that cause an organism to fluoresce and the like.

As used herein, “transfection” is the process of deliberatelyintroducing exogenous nucleic acids into eukaryotic cells;“transformation” is used to describe non-viral DNA transfer in bacteria,non-animal eukaryotic cells and plant cells; and “transduction” is usedto describe virus-mediated DNA transfer.

As used herein, a “reporter gene” is a gene placed under the control oftranscriptional regulatory elements and is engineered into a recombinantDNA construct and inserted into a cell or organism. Commonly usedreporter genes that induce visually-identifiable characteristics usuallyinvolve fluorescent proteins or enzymes that act on a substrate toproduce a luminescent product. Examples include the gene that encodesjellyfish GFP, the enzyme luciferase, and the red fluorescent proteinfrom the gene dsRed. The GUS gene has been commonly used in plants. Acommon reporter in bacteria is the LacZ gene, which encodes the proteinbeta-galactosidase. An example of a selectable-marker which is also areporter in bacteria is the chloramphenicol acetyltransferase (CAT)gene, which confers resistance to the antibiotic chloramphenicol. Thoughnot a gene per se, as used herein, the term “reporter gene” alsoincludes mRNA naturally transcribed by a cell; no visual or enzymaticmodifications are needed. Furthermore, as used herein, reporter gene isany modified or unmodified endogenous gene of a cell.

As used herein, a “reporter cell” is an engineered cell into which areporter gene has been inserted, or an un-engineered cell line,unmodified with an exogenously-added reporter gene, whose mRNAexpression changes upon the interaction of one or more ligands with oneor more molecules on the cell surface, or cells from primary culture.

As used herein, a “microelectromechanical systems device” or MEMS devicemeans an apparatus with components or features with relatively smalldimension between approximately 1 to 100 micrometers in size. The deviceenables miniaturization of biological assays such that single cellanalysis is possible.

As used herein, “microwell” refers to sub millimeter structures with avolume between approximately 1 picoliter and 500 nanoliters. Themicrowell is typically constructed in a shape that allows dense packingon a planar substrate, i.e., the shape is triangular, rectangular, orhexagonal. Microwells can be either opened by removing one surface,usually at the top, or closed by placing the top in contact with othersurfaces such as capture or support surfaces. The microwell can behomogeneous, or constructed out of dissimilar materials, including butnot limited to glass, photoresist, or polydimethylsiloxane (PDMS).

As used herein, “kinetic properties” refer to the rates of reactionk_(off), k_(on), and their ratio K_(D) between members of a proteincomplexes. For a binary protein complex, the dissociation constant K_(D)is monotonically related to the Gibbs free energy which describes thework obtainable from an isothermal, isobaric process, conditions closelyapproximated in living systems.

As used herein, a “target binding protein” or “target protein” is aprotein to which a biologically active agent of interest binds. Thetarget binding protein could be a protein that typically binds to thebiologically active agent of interest; for example, a cytokine, wherethe biologically active agent of interest is a receptor; or an antigen,where the biologically active agent of interest is an antibody.Alternatively, the target binding protein could be a protein that doesnot typically bind to the biologically active agent of interest; forexample, a control cytokine.

As used herein, “binding agent” or “capture agent” is a molecule used toimmobilize a biologically active agent. Capture agents can include butare not limited to oligonucleotides, DNA, RNA, protein, small molecules,peptides, aptamers, antibodies etc., which have an affinity for naturalor artificial ligands.

As used herein, a “solid surface” or “solid support” is any sort ofsurface or support. It may be made of glass, plastics, nitrocellulose,polyvinylidene fluoride, or other highly non-reactive materials. Abinding/capture agent may be attached (where the support may then bereferred to as “a capture surface” or “capture support”), in which casethe binding/capture agent may coat the solid surface, or may bedistributed in discrete locations on the solid surface, for example inspots, localized into pads, or configured into a line. A DNA microarrayis one of the many examples of a capture surface or capture support.

The term “monoclonal antibody” relates to an immunoglobulin made by asingle clone of an antibody-producing cell. All monoclonal antibodies ofthe same specificity are identical except for natural mutants thereof.The term “antibody” as used herein is understood to mean intactmolecules of immunoglobulins as well as fragments thereof (including butnot limited to Fab, F(ab′), Fv, scFv).

As used herein, a “ligand” is a substance that is able to bind to andform a complex with a cognate receptor molecule.

The term “B cell” is used herein to mean an immune cell that is highlyspecialized for making immunoglobulins. A B cell is a lymphocyte andprovides humoral immunity. A B cell produces an antibody that recognizesantigen molecules and can mature into a plasma cell. The term “plasmacell” is intended to mean a cell that develops from a B lymphocyte andcan secrete immunoglobulins at high rate. Throughout this applicationthe term “B cell” is intended to encompass “plasma cells” and viceversa. In general both are intended to encompass terms referring tocells which produce antibodies of interest.

Biologically active agents are characterized by their “binding affinity”to a given target biologically active agent, for example a protein. Forexample, an antibody is characterized by its affinity to a binding siteor epitope.

As used herein, a “receptor agonist” is a molecular entity that binds toa receptor of a cell and triggers a response by that cell; an “inverseagonist” or “antagonist” is an agent that binds to the same receptor asan agonist but induces a response opposite to that of an agonist; and a“receptor antagonist” is a type of molecule that binds to a receptor butdoes not provoke a response but blocks or dampens other agonist-mediatedresponses. A “co-agonist” is any of a number of molecules that worktogether to form an agonist. Throughout this application the term“receptor agonist” is intended to also encompass “superagonist”, “fullagonist”, “partial agonist”, “partial inverse agonist”, “full inverseagonist”, “co-agonist”, “allosteric agonist” or any other variants ofagonist.

The term “receptor antagonist” is intended to encompass “competitiveantagonist”, “non-competitive antagonist”, “uncompetitive antagonist”,“silent antagonist” or any other variants of antagonist.

The term “modulator” is intended to encompass an agent modulating theactivity of a naturally-occurring receptor agonist or receptorantagonist.

The term “random code” is used herein to mean a randomly generated DNAsequence of any length (i.e. 0 or more nucleotides) derived from anoligonucleotide with mixed bases (Ns) at strategic positions. Thesemixed bases can be consecutive or demarcated by specific bases (e.g.NNNGNNCNN). A random code of one or more nucleotides in combination withtag(s) enables counting of sequenced PCR amplified cDNA moleculesgenerated from independent mRNA molecules to yield the Absolute CopyNumber of mRNA of a reporter gene in a reporter cell. In circumstanceswhere the random code is not needed it is included herein with a lengthof 0 nucleotides.

As used herein, the cDNA molecules having a random code incorporated are“coded cDNA” molecules.

As used herein, the term “Next Generation Sequencing” or NGS means a DNAsequencing technology that analyzes in a massively parallel mannersingle DNA molecule or a clonally amplified population of DNA molecules.Current examples of companies marketing such a technology are Roche 454,Ion Torrent from Life Technologies, and Illumina. Often millions ofsequences are produced in a single run, compared to orders of magnitudefewer sequences produced by older technologies such as Sangersequencing. The number of high quality nucleotides sequenced varies from30 to 500 nucleotides. Each company's offering requires slightlydifferent protocols to prepare samples and they consume differentamounts of DNA.

As used herein, the term “a feature or features” means a microscopic DNAspot with a cluster of oligonucleotides attached to a defined locationof the solid surface of a DNA microarray.

As used herein, “similar sequences” means nucleic acid sequences fromthe same molecule displaying the identical sequence or a sequence withnucleotide errors introduced by sample preparation or DNA sequencingmethodology.

As used herein, “original molecule” means the molecule present in asample population of nucleic acids prior to PCR amplification, not amolecule in that has been amplified from the sample population ofnucleic acids.

As used herein, aggregating means to combine similar sequences with thesame random code into a single sequence representing the nucleic acidsequence of the original molecule.

DETAILED DESCRIPTION OF THE INVENTION

This invention generally relates to methods, devices and kits forscreening a plurality of secreting cells for functional activity bymeasuring the NCN of mRNA from one or more reporter genes produced byone or more types of reporter cells in response to secreted moleculesacting as a receptor agonist, a receptor antagonist, or a modulator. Inone embodiment, a plurality of secreting cells are placed in a pluralityof microwells with a majority of microwells containing a singlesecreting cell. One or more types of reporter cells are also placed inthe microwells. Secreted molecules by the secreting cells are capturedon one or more support surfaces brought into contact with themicrowells, at the same time, the secreted molecules are allowed tointeract with the receptor on the reporter cells in the microwells as areceptor agonist or receptor antagonist. Subsequently the secretingcells and reporter cells are lysed releasing their mRNA. The mRNAencoding the secreted molecules, the mRNA from one or more reportergenes in one or more types of reporter cells, and housekeeping gene(s)from each type of reporter cells are captured on an oligonucleotidecapture support containing mRNA capture oligos comprising a uniquenucleotide tag and a random code at each feature on the oligonucleotidecapture support corresponding to each microwell location. The capturedmRNA on the oligonucleotide capture support is converted into cDNAincorporating the tags and optionally the random code, and tagged andcoded cDNA is sequenced using NGS technology. The kinetic properties ofthe secreted molecules to one or more specific moieties are measured.The mRNA sequence encoding the secreted molecules is associated with themeasured kinetic properties and Absolute Copy Number (ACN) of the mRNAof one or more reporter genes from each type of reporter cells isdetermined. The ACN of mRNA of housekeeping gene(s) of each type ofreporter cells is determined and used to calculate the Normalized CopyNumber (NCN) of mRNA of one or more reporter genes from each type ofreporter cells. The NCN for a given secreted molecule is assessed andused in a manner analogous to the reporter cells currently sold and iswell known to those skilled in the art.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular methods anddevices described, and as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell or sequence” may include a plurality of such cells or sequences andreference to “the well or addresses” may include reference to one ormore wells or addresses and equivalents thereof known to those skilledin the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

The current invention is envisaged as one or more methods, one or morephysical devices, and/or one or more kits, each covering the entirety ora portion of the invention. Screening means searching through acollection of many cells. The collection can contain many copies of thesame cell, many copies of different cells, or both: many copies of manydifferent cells.

Previous applications filed by the present inventors teach a method formeasuring the kinetics of a molecule secreted from a secreting cell anddetermining the sequence of the mRNA coding for the secreted molecule.The present invention provides an improvement to these methods byteaching methods to perform tests for the functional activity of thesecreted molecule. Functional activity generally refers to themodification of intracellular signaling due to the increase or decreaseof binding of a ligand at a cellular receptor. Functional activity isdistinct from antigen-antibody binding because, when eliciting an immuneresponse from a host, an antigen must be a form convenient for hostexposure as an immunogen. The form often may be distinct from the formactive in a therapeutic situation. Furthermore, the host's focus uponintroduction of foreign material is to identify and eliminate thepathogen, and one epitope is often as good as another. In a therapeuticsituation, only specific epitopes may be useful for treating ormodifying a disease or symptoms. Therefore, it is advantageous to test,for example, antibody-antigen interactions as close as possible to thesituation presented in the therapeutic environment. Functional activityof, e.g., an antibody is much closer to a therapeutic situation, and afunctional assay tests for functional activity. Such a functional assayis presented, e.g., in Example 1, where an un-engineered reporter cellline (HeLa) is used for identifying a TNF-α blocker, which can be usefulin treating a variety of autoimmune diseases including psoriasis,employing the present invention.

U.S. Pat. No. 8,309,317 (Chen et al., Nov. 13, 2012), U.S. Pat. No.8,309,035 (Chen et al., Nov. 13, 2012), and US Pub. No. 20110190148(Chen et al., Aug. 4, 2011), each of which are incorporated herein forall purposes, describe a MEMS device that is used in the presentinvention. Briefly, microwells are fabricated into an elastomericmaterial on a MEMS device such that thousands of microwells fit in thearea of a microscope slide. The microwells are open at the top.Secreting cells are dispersed from the top of the MEMS device and sinkinto the microwells after short period of time. The number of thesecreting cells dispersed from the top of the MEMS device is less thanthe number of the wells so that, on average, a single cell occupies asingle well.

Secreted molecules, such as antibodies produced in certain immune cells,e.g., plasma cells, are secreted at a high rate—often more than athousand molecules per second. In microwells (having volumes of, e.g.,180 pL) such a rate results in a high concentration (approximately 1 nM)of secreted molecules in a short period of time (less than one minute).Sequential capture surfaces may be brought into contact with thesecreting cells in the microwells in order to capture the secretedmolecules from the secreting cells in the microwells. For example, asolid support coated with an antibody capture agent (e.g., Protein G,Protein A, etc.) may be used to contact the surface of the MEMS deviceto capture antibodies secreted by the plasma cell(s) contained in eachmicrowell. Second, third, and so forth, similar antibody capturesurfaces can be used sequentially to capture additional secretedantibodies, each capture resulting in an addressable antibody arrayreflecting the locations of all the plasma cells contained in themicrowells. Each slide can be, for example, reacted with increasingconcentrations of a fluorescently labeled antigen or a fluorescentlylabeled molecule related to the antigen in order to compute estimates ofthe dissociation constants and k_(off). The specifics of kineticsmeasurements are further elaborated in U.S. Pat. No. 8,309,317 (Chen etal., Nov. 13, 2012), U.S. Pat. No. 8,309,035 (Chen et al., Nov. 13,2012), and US Pub. No. 20110190148 (Chen et al., Aug. 4, 2011), whichare incorporated herein in their entirety.

In addition, the secreting cells optionally may be lysed and theirnucleic acid content (including mRNA) released. Messenger RNA encodingthe secreted molecules from the secreting cells can be captured on anoligonucleotide capture support containing mRNA capture oligoscomprising a unique nucleotide tag and a random code at each featurecorrelate to the position of a microwell. Captured mRNA on theoligonucleotide capture support is converted to cDNA incorporating thetags and optionally random code, and the tagged and coded cDNA issequenced using NGS technology. Because the tags are unique to eachlocation of the oligonucleotide capture support, the sequence of thetagged cDNAs can be related back to the microwell from which it wascaptured. Thus, the binding kinetics properties obtained from anantibody associated with a small area of the capture surface derivedfrom a secreting cell contained in each microwell can be associated withthe mRNA sequence of, e.g., both the light and heavy chains of anantibody. In addition, each cDNA sequence containing a distinct randomcode will be counted as an independent molecule. The count of theindependent cDNA molecules is a good proxy of the responding or steadystate levels of the mRNA at the time of cell lysis. As such,implementation of location-specific tags and molecule-specific randomcodes enables a parallel transcription analysis of the secreting cellsand reporter cells by NGS to screen for functional activity of thesecreted molecules (e.g., antibodies) as receptor agonists, receptorantagonists, or modulators. The specifics of mRNA sequencing and theassociation of sequencing with kinetics are further elaborated in U.S.Pat. No. 8,309,317 (Chen et al., Nov. 13, 2012), U.S. Pat. No. 8,309,035(Chen et al., Nov. 13, 2012), and US Pub. No. 20110190148 (Chen et al.,Aug. 4, 2011).

The present invention uses some of the processes described in U.S. Pat.No. 8,309,317 (Chen et al., Nov. 13, 2012), U.S. Pat. No. 8,309,035(Chen et al., Nov. 13, 2012), and US Pub. No. 20110190148 (Chen et al.,Aug. 4, 2011), in novel ways. In one embodiment, one or more reportercells are deposited in the microwells of a MEMS device. Theconcentration of reporter cells is such that nearly every microwell isoccupied by the reporter cells. By contrast, to maximize the chance ofdepositing a single secreting cell in a microwell, the concentration ofthe secreting cell is lower than that of the reporter cells; thereforethere will be microwells unoccupied by the secreting cells. The reportercells may be designed to produce readily assayable proteins in responseto specific binding activity; alternatively, reporter cells that requireno engineering may be used and changes in expression of endogenous genesin response to a specific binding activity may be adequate to reportperturbation of the signal transduction event of a targeted pathway by asecreted molecule produced by a secreting cell.

An example of such an assay is screening of mouse plasma cells, a typeof secreting cell secreting an antibody to an antigen (e.g., humanTNF-α) that interacts with an endogenous receptor on one or morereporter cells; e.g., human embryonic kidney 293 cell line with anengineered reporter gene such as luciferase driven by a promoterregulated by a nuclear factor kappa B (NF-κB) response element tomonitor the NF-κB signal transduction pathways, or HeLa (humanepithelial carcinoma cell line) with the endogenous interleukin-6 geneas the reporter gene. Upon binding by human TNF-α to its cognatereceptor on the surface of a reporter cell, endogenous NF-κBtranscription factors are immobilized and bind to the DNA responseelements inducing transcription of the downstream responding genes,including the reporter gene. The objective is to screen for a mouseantibody that will bind to human TNF-α and block TNF-α from interactingwith its receptor to block activation of the NF-κB pathway. This is anexample of screening mouse antibodies for a modulator.

In the present invention, the protein product (e.g., luciferase) is notmeasured; instead, the ACN of the responding mRNA levels of one or morereporter genes as well as optionally the ACN of the steady state mRNAlevels from one or more housekeeping genes are measured by sequencingthe tagged and optionally coded cDNA molecules corresponding to themRNAs and counting the occurrence thereof. The present inventionprovides greater flexibility in the design of reporter cells, reducesthe time required to measure the changes in the expression of a reportergene, tremendously increases throughput by miniaturizing andparallelizing the assay, and eliminates the toxic (and often costly)chemicals used in reporter assays. With the ACNs of the mRNA levels ofone or more reporter genes and ACNs of the mRNA of one or morehousekeeping genes of the reporter cells measured, the NCN for the mRNAof a given reporter gene can be calculated. To ensure validity of theNCN, it is imperative that the secreting cells and reporter cells are ofdifferent origin (e.g., mouse plasma cells as secreting cells andengineered human HEK239 cell line or un-engineered HeLa cells (human) asreporter cells) such that the sequence of the housekeeping gene(s) usedin determining the NCNs of the reporter gene in reporter cells isdistinguishable from that of the housekeeping gene(s) of the secretingcells.

By comparing the NCN of reporter gene(s) of the reporter cells in thosemicrowells unoccupied by secreting cells and the NCN of the reportergene(s) from the reporter cells in those microwells containing secretingcells, one may assess the ability of a mouse plasma cell (e.g., asecreting cell) secreting an antibody to block the activity of humanTNF-α. At the same time, the cDNA sequence of the variable domain forboth the heavy and light chains of the antibody is determined. Thisvariable domain sequence for both chains can then be used to generate arenewable source of the monoclonal antibody for further evaluation.

Anatomy of Two Exemplary and Adjacent Features with their mRNA CaptureProbes on an Area of a DNA Microarray.

In this example, one feature will mate with a microwell containing oneor more reporter cells. FIG. 1 shows a solid support 104 with twofeatures, 103 a and 103 b, each consisting, for illustration purposes,of 5 oligonucleotides with the DNA sequence from the 5′ end on the leftand the 3′ end on the right. In reality, there can be millions ofoligonucleotides contained within each feature on a DNA microarray. Attime point 107 the oligonucleotides are unoccupied and available tocapture mRNA released from lysed cells. Along the length of eacholigonucleotide, starting from the 5′ end and moving toward the 3′ end,there are 4 Ts serving as a spacer; tag regions 101 a(b) represented bybases in bold type unique to each feature identifying the capturelocation; optional random code region 102 a(b) represented by lower caseletters unique to each oligonucleotide and allows the cDNA molecules tobe accurately associated with the cognate independently-captured mRNAmolecules. The random codes provide a numerical basis to correct anybias introduced by PCR amplification. Finally, there is mRNA captureregion 100 which, in this example, can be one of two differentsequences; one sequence can capture mRNA from a reporter gene and theother sequence can capture mRNA from a housekeeping gene.

At time point 108, the oligonucleotides on feature 103 a(b) capture mRNA105 a 1(b 1) represented by heavy waved lines from the reporter gene andmRNA 105 a 2(b 2) represented by thin waved lines from the housekeepinggene. These oligonucleotides serve to prime cDNA synthesis using thecaptured mRNA as a template. Those oligonucleotides capturing mRNA fromthe reporter gene yield cDNA 106 a 1(b 1) represented by a thin brokenline and those capturing mRNA from the housekeeping gene yield cDNA 106a 2(b 2) represented by a thick broken line. Once cDNA 106 a 1(b 1) isamplified and sequenced by NGS technology, the mRNA capture location canbe computed using the tag and the number of independent mRNAscaptured—proportional to the levels of mRNA within the reporter cellsand defined herein as the ACN of the reporter gene mRNA—can be inferredfrom the number of cDNA sequences bearing the same tag and a uniquerandom code. The use of the random code enables correction of any biasin the cDNA molecule count introduced by PCR amplification. Similarly,cDNA 106 a 2(b 2) can be counted to yield the ACN of the housekeepinggene mRNA of cells in the microwell mated to the second exemplaryfeature. Subsequently, the ratio of the ACN of the reporter gene mRNAand the ACN of the housekeeping gene mRNA defined herein as the NCN ofthe reporter gene mRNA from reporter cells in different microwells canbe determined. The NCN as used herein assesses the activity of asecreted molecule by its effect on transcriptional changes of thereporter gene in reporter cells illustrated in various embodiments.

The following particular embodiments are examples of this invention andare not meant to exhaustively describe its application.

Embodiment 1—Detection of an Receptor Agonist

The methods of the present invention are used in certain embodiments todetect a secreted molecule that acts as a receptor agonist to a receptoron a reporter cell. Referring to FIG. 2, two adjacent microwells, 200 aand 200 b, viewed from the side of a MEMS device, are drawn at varioustime points 200, 201, 202, and 203. Multiple reporter cells 206 withcell surface receptors 214 are deposited in microwells 200 a(b). Oncereporter cells 206 have settled into each microwell, a single secretingcell 207 a(b) is deposited into microwell 200 a(b). Secreting cell 207a(b) secretes monoclonal antibody 208 a(b) at time point 202. Monoclonalantibody 208 a(b) diffuses throughout microwell 200 a(b). Monoclonalantibody 208 a binds to cell surface receptor 214 on reporter cell 206and such a binding event is denoted as 209 at time point 202. Also attime point 202, monoclonal antibody 208 b in microwell 200 b does notbind to the cell surface receptors 214 and remains freely diffusing inmicrowell 200 b. In between time points 202 and 203 a lysing agent isdispensed into microwells 200 a(b) disrupting cells in the microwellsand causing the release of the cells' mRNA content. Immediatelyfollowing lysis, at time point 203, an oligonucleotide capture support204 containing 2 features consist of a population of captureoligonucleotides 205 a(b) complementary to mRNA for, e.g., the heavychain and light chain of the secreted antibody 208 a(b) secreted bysecreting cells 207 a(b), the reporter gene(s) and the housekeepinggene(s) expressed in reporter cells 206 is brought into close proximityof microwells 200 a(b) severely retarding diffusion of the mRNA out ofthe microwells. Alternatively, messenger RNA 210 a(b) represented bysuper heavy waved lines for the heavy chain of the secreted molecule 208a(b), and mRNA 211 a(b) represented by medium waved lines for the lightchain of the secreted molecule 208 a(b) from secreting cell 207 a(b)diffuse and are captured by the appropriate capture oligonucleotides 205a(b). At the same time, the reporter gene mRNA 212 a(b) represented bythin waved lines and the housekeeping gene mRNA 213 a(b) represented bydotted lines from the reporter cells 206 in microwell 100 a(b) diffuseand are captured by the appropriate capture probes 205 a(b).

Oligonucleotide probes 205 a(b) each contain a tag and an optionalrandom code as detailed in FIG. 1. When cDNA molecules are synthesizedfrom mRNA primed with the oligonucleotide probes 205 a(b), everysequence with the same tag most likely will have incorporated adifferent random code. When tagged and coded cDNA is sequenced usingNGS, those molecules with the same tags are collated and the random codeis examined. As such, the random codes provide a direct way to count theoriginal distribution of cDNA molecules reflecting the captured mRNAmolecules and to calculate the ACN defined herein for the reporter genemRNA 212 a(b) despite any potential bias introduced, e.g., by PCRamplification of tagged and coded cDNA. The amount of captured mRNAmolecules reflects the mRNA level of the responding reporter gene aswell as that of the constitutive housekeeping gene. Similarly, the ACNfor the housekeeping gene mRNA 213 a(b) is determined.

In preferred embodiments, the ratio of the ACN for the reporter genemRNA 212 a(b) and ACN for the housekeeping gene mRNA 213 a(b) iscomputed to yield the NCN of the reporter gene mRNA 212 a(b) of thereporter cell 206 in a microwell whose location is delineated by the tagembedded in the cDNA molecules. The NCN for the reporter gene mRNA 212 aof reporter cell 206 in microwell 200 a—given the secreted molecule 208a acting as a receptor agonist—will increase compared to a) the NCN ofthe reporter gene mRNA 212 b in microwell 200 b containing the secretingcell 207 b secreting a non-functional secreted molecule 208 b that doesnot display a binding event similar to 209; or to b) the NCN of thereporter gene mRNA of the reporter cell in microwells containing nosecreting cells.

Embodiment 2—Detection of an Inverse Agonist, a Type of Receptor Agonist

The methods of the present invention are used in certain embodiments todetect a secreted molecule that acts as a receptor agonist to a receptoron a reporter cell. Referring to FIG. 3, two adjacent microwells, 300 aand 300 b, viewed from the side of a MEMS device, are drawn at varioustime points 300, 301, 302, and 303. Multiple reporter cells 306 withcell surface receptors 314 are deposited in microwells 300 a(b). Oncereporter cells 306 have settled into each microwell, a single secretingcell 307 a(b) is deposited into microwell 300 a(b). Secreting cell 307a(b) secretes monoclonal antibody 308 a(b) at time point 302. Monoclonalantibody 308 a(b) diffuses throughout microwell 300 a(b). Monoclonalantibody 308 a binds to cell surface receptor 314 on reporter cell 306and such a binding event is denoted as 309 at time point 302. Also attime point 302, monoclonal antibody 308 b in microwell 300 b does notbind to the cell surface receptors 314 and remain freely diffusing inmicrowell 300 b. In between time points 302 and 303 a lysing agent isdispensed into microwells 300 a(b) disrupting cells in the microwellsand causing the release of the cells' mRNA content. Immediatelyfollowing lysis, at time point 303, an oligonucleotide capture support304 containing 2 features consist of a population of captureoligonucleotides 305 a(b) complementary to mRNA for the heavy chain andlight chain of the secreted antibody 308 a(b) secreted by secretingcells 307 a(b), the reporter gene(s) and the housekeeping gene(s)expressed in reporter cells 306 is brought into close proximity ofmicrowells 300 a(b) severely retarding diffusion of the mRNA out of themicrowells. Alternatively, messenger RNA 310 a(b) represented by superheavy waved lines for the heavy chain of the secreted molecule 308 a(b),and mRNA 311 a(b) represented by medium waved lines for the light chainof the secreted molecule 308 a(b) from secreting cell 307 a(b) diffuseand are captured by the appropriate capture oligonucleotides 305 a(b).At the same time, mRNA 312 a(b) represented by thin waved lines for thereporter gene and mRNA 313 a(b) represented by dotted lines for thehousekeeping gene from the reporter cells 306 in microwell 300 a(b)diffuse and are captured by the appropriate capture probes 305 a(b).

Oligonucleotide probes 305 a(b) each contain a tag and an optionalrandom code as detailed in FIG. 1. When cDNA molecules are synthesizedfrom mRNA primed with the oligonucleotide probes 305 a(b), everysequence with the same tag most likely will have incorporated adifferent random code. When tagged and coded cDNA is sequenced usingNGS, those molecules with the same tags are collated and the random codeis examined. As such, the random codes provide a direct way to count theoriginal distribution of cDNA molecules reflecting the captured mRNAmolecules and to calculate the ACN defined herein for the reporter genemRNA 312 a(b) despite any potential bias introduced by PCR amplificationof tagged and coded cDNA. The amount of captured mRNA molecules reflectsthe mRNA level of the responding reporter gene as well as that of theconstitutive housekeeping gene. Similarly, the ACN for the housekeepinggene mRNA 313 a(b) is determined.

In preferred embodiments, the ratio of the ACN for the reporter genemRNA 312 a(b) and ACN for the housekeeping gene mRNA 313 a(b) iscomputed to yield the NCN of the reporter gene mRNA 312 a(b) of thereporter cell 306 in a microwell whose location is delineated by the tagembedded in the cDNA molecules. The NCN for the reporter gene mRNA 312 aof reporter cell 306 in microwell 300 a—given the secreted molecule 308a acting as an inverse agonist—a type of receptor agonist, will decreasecompared to a) the NCN of the reporter gene mRNA 312 b in microwell 300b containing the secreting cell 307 b secreting a non-functionalsecreted molecule 308 b that does not display a binding event similar to309; or to b) the NCN of the reporter gene mRNA of the reporter cell inmicrowells containing no secreting cells.

Embodiment 3—Detection of a Modulator

The methods of the present invention are used in certain embodiments todetect a secreted molecule that acts as a receptor agonist to a receptoron a reporter cell. Referring to FIG. 4, two adjacent microwells, 400 aand 400 b, viewed from the side of a MEMS device, are drawn at varioustime points 400, 401, 402, and 403. Multiple reporter cells 406 withcell surface receptors 414 are deposited in microwells 400 a(b). Oncereporter cells 406 have settled into each microwell, a single secretingcell 407 a(b) is deposited into microwell 400 a(b). Secreting cell 407a(b) secretes monoclonal antibody 408 a(b) at time point 402. Monoclonalantibody 408 a(b) diffuses throughout microwell 400 a(b). At time point402, a ligand 415 to the cognate receptor 414 on reporter cells 406 isintroduced and is diffusing throughout microwell 400 a(b). Monoclonalantibody 408 a binds to the ligand 415 and such a binding event isdenoted as 409 at time point 402. Also at time point 402, monoclonalantibody 408 b in microwell 400 b does not bind to the ligand 415 andremain freely diffusing in microwell 400 b resulting in binding of theligand 415 to the cognate receptor 414. In between time points 402 and403 a lysing agent is dispensed into microwells 400 a(b) disruptingcells in the microwells and causing the release of the cells' mRNAcontent. Immediately following lysis, at time point 403, anoligonucleotide capture support 404 containing 2 features consist of apopulation of capture oligonucleotides 405 a(b) complementary to mRNAfor the heavy chain and light chain of the secreted antibody 408 a(b)secreted by secreting cells 407 a(b), the reporter gene(s) and thehousekeeping gene(s) expressed in reporter cells 406 is brought intoclose proximity of microwells 400 a(b) severely retarding diffusion ofthe mRNA out of the microwells. Alternatively, messenger RNA 410 a(b)represented by super heavy waved lines for the heavy chain of thesecreted molecule 408 a(b), and mRNA 411 a(b) represented by mediumwaved lines for the light chain of the secreted molecule 408 a(b) fromsecreting cell 407 a(b) diffuse and are captured by the appropriatecapture oligonucleotides 405 a(b). At the same time, mRNA 412 a(b)represented by thin waved lines for the reporter gene and mRNA 413 a(b)represented by dotted lines for the housekeeping gene from the reportercells 406 in microwell 400 a(b) diffuse and are captured by theappropriate capture probes 405 a(b).

Oligonucleotide probes 405 a(b) each contain a tag and an optionalrandom code as detailed in FIG. 1. When cDNA molecules are synthesizedfrom mRNA primed with the oligonucleotide probes 405 a(b), everysequence with the same tag most likely will have incorporated adifferent random code. When tagged and coded cDNA is sequenced usingNGS, those molecules with the same tags are collated and the random codeis examined. As such, the random codes provide a direct way to count theoriginal distribution of cDNA molecules reflecting the captured mRNAmolecules and to calculate the ACN defined herein for the reporter genemRNA 412 a(b) despite any potential bias introduced by PCR amplificationof tagged and coded cDNA. The amount of captured mRNA molecules reflectsthe mRNA level of the responding reporter gene as well as that of theconstitutive housekeeping gene. Similarly, the ACN for the housekeepinggene mRNA 413 a(b) is determined.

In preferred embodiments, the ratio of the ACN for the reporter genemRNA 412 a(b) and ACN for the housekeeping gene mRNA 413 a(b) iscomputed to yield the NCN of the reporter gene mRNA 412 a(b) of thereporter cell 406 in a microwell whose location is delineated by the tagembedded in the cDNA molecules. The NCN for the reporter gene mRNA 412 aof reporter cell 406 in microwell 400 a—given the secreted molecule 408a acting as a modulator—will decrease compared to a) the NCN of thereporter gene mRNA 412 b in microwell 400 b containing the secretingcell 407 b secreting a non-functional secreted molecule 408 b that doesnot display a binding event similar to 409; or to b) the NCN of thereporter gene mRNA of the reporter cell in microwells containing nosecreting cells.

Embodiment 4—Detection of a Receptor Antagonist

Yet another embodiment of the present invention is used to detect asecreted molecule that acts as a receptor antagonist to a receptor on areporter cell. Referring to FIG. 5, two adjacent microwells, 500 a and500 b, viewed from the side of a MEMS device, are drawn at various timepoints 500, 501, 502, and 503. Multiple reporter cells 506 with cellsurface receptors 514 are deposited in microwells 500 a(b). Oncereporter cells 506 have settled into each microwell, a single secretingcell 507 a(b) is deposited into microwell 500 a(b). Secreting cell 507a(b) secretes monoclonal antibody 508 a(b) at time point 502. Monoclonalantibody 508 a(b) diffuses throughout microwell 500 a(b). At time point502, a ligand 515 to the receptor 514 on reporter cells 506 isintroduced and is diffusing throughout microwell 500 a(b). Monoclonalantibody 508 a binds to the receptor 514 and such a binding event isdenoted as 509 at time point 502. Also at time point 502, monoclonalantibody 508 b in microwell 500 b does not bind to the receptor 514 andremain freely diffusing in microwell 500 b resulting in binding of theligand 515 to the receptor 514. In between time points 502 and 503 alysing agent is dispensed into microwells 500 a(b) disrupting cells inthe microwells and causing the release of the cells' mRNA content.Immediately following lysis, at time point 503, an oligonucleotidecapture support 504 containing 2 features consist of a population ofcapture oligonucleotides 505 a(b) complementary to mRNA for the heavychain and light chain of the secreted antibody 508 a(b) secreted bysecreting cells 507 a(b), the reporter gene(s) and the housekeepinggene(s) expressed in reporter cells 506 is brought into close proximityof microwells 500 a(b) severely retarding diffusion of the mRNA out ofthe microwells. Alternatively, messenger RNA 510 a(b) represented bysuper heavy waved lines for the heavy chain of the secreted molecule 508a(b), and mRNA 511 a(b) represented by medium waved lines for the lightchain of the secreted molecule 508 a(b) from secreting cell 507 a(b)diffuse and are captured by the appropriate capture oligonucleotides 505a(b). At the same time, the reporter gene mRNA 512 a(b) represented bythin waved lines and the housekeeping gene mRNA 513 a(b) represented bydotted lines from the reporter cells 506 in microwell 500 a(b) diffuseand are captured by the appropriate capture probes 505 a(b).

Oligonucleotide probes 505 a(b) each contain a tag and an optionalrandom code as detailed in FIG. 1. When cDNA molecules are synthesizedfrom mRNA primed with the oligonucleotide probes 505 a(b), everysequence with the same tag most likely will have incorporated adifferent random code. When tagged and coded cDNA is sequenced usingNGS, those molecules with the same tags are collated and the random codeis examined. As such, the random codes provide a direct way to count theoriginal distribution of cDNA molecules reflecting the captured mRNAmolecules and to calculate the ACN defined herein for the reporter genemRNA 512 a(b) despite any potential bias introduced by PCR amplificationof tagged and coded cDNA. The amount of captured mRNA molecules reflectsthe mRNA level of the responding reporter gene as well as that of theconstitutive housekeeping gene. Similarly, the ACN for the housekeepinggene mRNA 513 a(b) is determined.

In preferred embodiments, the ratio of the ACN for the reporter genemRNA 512 a(b) and ACN for the housekeeping gene mRNA 513 a(b) iscomputed to yield the NCN of the reporter gene mRNA 512 a(b) of thereporter cell 506 in a microwell whose location is delineated by the tagembedded in the cDNA molecules. The NCN for the reporter gene mRNA 512 aof reporter cell 506 in microwell 500 a—given the secreted molecule 508a acting as a receptor antagonist—will decrease compared to a) the NCNof the reporter gene mRNA 512 b in microwell 500 b containing thesecreting cell 507 b secreting a non-functional secreted molecule 508 bthat does not display a binding event similar to 509; or b) the NCN forthe reporter gene mRNA 512 a of reporter cell 506 in microwell 500 awill roughly equal the NCN of the reporter gene mRNA of the reportercell in microwells containing no secreting cells.

EXAMPLE

The following example is put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and is not intended to limit thescope of what the inventors regard as their invention nor is it intendedto represent that the experiment below is all or the only experimentthat could be performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1: Identification of a Murine Anti-Human TNF-α AntibodyFunctioning as a Modulator

Immunization & Single Cell Suspension Generation.

Recombinant human TNF-α (rhTNF-α, Insight Genomics, Falls Church, Va.)is used to immunize young BALB/c mice with 50 μg rhTNF-α in Freund'scomplete adjuvant (Sigma-Aldrich, St. Louis, Mo.) by intraperitonealinjection (i.p.) on day 0. Fifty μg rhTNF-α in incomplete Freund'sadjuvant (IFA, Sigma-Aldrich, St. Louis, Mo.) is then administered bysubcutaneous injection (s.c.) on day 14 and on day 28, serum iscollected on day 36 for titration, and 50 μg rhTNF-α in IFA isadministered by s.c. on day 42, with a final boost with 100 μg rhTNF-α(intravenously only) on day 56. The spleen, along with serum, isharvested on day 59. A single cell suspension of the splenocytes isgenerated by disrupting the tissue via mashing between two frostedmicroscope slides using 10 mL of RPMI medium (ATCC, Manassas, Va.). Thesuspension is filtered through a 70 μm mesh (BD Biosciences, San Jose,Calif.) to remove clumps.

CD138+ Plasma Cell Isolation, Antibody Capture, and AntigenInterrogation.

Freshly isolated splenocytes from the above hyperimmunized mice arefurther processed using a commercial kit to enrich for plasma cellsbased on cell surface expression of CD138 (Miltenyi, Auburn, Calif.).Human cervical epithelial cells HeLa are used as the reporter cell andits endogenous Interleukin-6 gene as the reporter gene (ATCC, Manassas,Va.). HeLa cells are first spread at a concentration on a PDMS devicesuch that deposition of 3 to 10 cells per microwells is favored.Afterwards, freshly enriched plasma cells are then spread on the samePDMS device at a lower cell concentration to favor deposition of asingle cell per microwell. Antibody secreted from each plasma cell iscaptured on a derivatized microscope slide as described in U.S. Pat. No.8,309,317 (Chen et al., Nov. 13, 2012), U.S. Pat. No. 8,309,035 (Chen etal., Nov. 13, 2012), and US Pub. No. 20110190148 (Chen et al., Aug. 4,2011). Antigen-specific antibody secreting cells are identified byinterrogating the antibody capture slide with increasing concentrationsof fluorescently labeled rhTNF-α.

TNF Stimulation and mRNA Capture.

After antibody capture, the medium on the PDMS is exchanged with thesame medium containing rhTNF-α at 20 ng/mL and cultured at 37° C. and 5%CO2 to allow stimulation of the reporter cell present in each microwell.For those microwells containing a plasma cell secreting an antibody thatfunctions as a modulator, rhTNF-α will be bound by the antibody and notreadily interact with the cognate receptor to activate the NF-kB signaltransduction pathway in HeLa cells (reporter cells) present in the samemicrowell. After 30 minutes of incubation with rhTNF-α, the medium isremoved and replaced with lysis buffer followed by prompt closure of thetop of the microwells with a custom oligonucleotide capture support(NimbleGen, Madison, Wis.), as described in U.S. Pat. No. 8,309,317(Chen et al., Nov. 13, 2012), U.S. Pat. No. 8,309,035 (Chen et al., Nov.13, 2012), and US Pub. No. 20110190148 (Chen et al., Aug. 4, 2011). Thecustom oligonucleotide capture support is prepared such that eachfeature contains capture probes for mRNAs for all subclasses (1, 2a, 2b,and 3) of the murine IgG heavy chain gene, the murine Ig kappa lightchain gene, and the murine Ig lambda light chain gene. Additionally, theoligonucleotide capture probes are expanded for the present invention toinclude the human Interleukin-6 (IL-6) gene (reporter gene), and thehuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene(housekeeping gene). Hybridization is allowed to proceed overnight. Inaddition, each oligonucleotide capture probe contains a unique tagspecifying the coordinate in relation to each microwell and an optionalrandom code for facilitating counting of independent cDNA fragmentssequenced with NGS to measure the independently captured mRNA molecules,which reflect the responding level of reporter gene mRNA and theconstitutive level of housekeeping gene mRNA.

cDNA Synthesis, PCR Amplification, and NGS.

Captured mRNA on the oligonucleotide capture support are converted intocDNA. A unique tag and an optional random code is incorporated into eachcDNA to allow for matching of mRNA to microwells and measurement of theindependently captured mRNA. The tagged and coded cDNA fragments of thefollowing mRNA molecules are then amplified: variable domain of IgGheavy chain subclasses, variable domain of Ig kappa light chain,variable domain of lambda light chain, a fragment of the humaninterleukin-6 gene, and a fragment of the human GAPDH gene. Theamplicons are then sequenced by NGS on a MiSeq (Illumina, San Diego,Calif.) with the 2×250 bp chemistry.

Bioinformatic Analysis of Images and DNA Sequences.

All fluorescent images are scanned at 532 nm on an Axon 4000A (10 micronresolution) and the files are processed using Mathematica 8.0 (WolframResearch). Three images of every slide are scanned at variousphotomultiplier voltages. Image files are processed in proprietaryMathematica algorithms in order to identify a) small areas with shapesof the microwells; b) signal intensities that are statisticallysignificant; and c) consistent baseline values.

Intensity values for each of 3 reaction concentrations (10 pM, 100 pM, 1nM) and 3 additional time points after wash are recorded. Concentrationvalues are used to estimate the K_(D) and signal amplitude that minimizethe squared sum of the difference between the expected and measuredsignals, commonly termed the Chisquared,

$\begin{matrix}{\chi^{2} = {\sum\limits_{i = 1}^{3}\; \left( {{Signal}_{i} - \frac{{Amp}\; {Conc}_{i}}{{Conc}_{i} + K_{D}}} \right)^{2}}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Bioinformatic Analysis of Images and DNA Sequences.

Signal_(i) is the measured digital value (0-65535), Conc_(i) is one of{10 pM, 100 pM, 1 nM}, and Amp and K_(D) are parameters varied until aminimum is reached. k_(off) is estimated from the 1 nM signal plus thethree ensuing washes by minimizing a similar difference between theexpected and measured signals:

$\begin{matrix}{\chi^{2} = {\sum\limits_{i = 1}^{4}\; \left( {{Signal}_{i} - {{Amp}\; e^{{- k}\mspace{14mu} {Time}_{i}}}} \right)^{2}}} & {{Equation}\mspace{14mu} 2}\end{matrix}$

Signal_(i) is the measured digital value (0-65535), Time_(i) is one of{5 min, 15 min, 45 min or 105 min}, and Amp and k are parameters varieduntil a minimum is reached. k is commonly called k_(off), measured insec-1, and is the reciprocal of the time needed for 63% of the boundantigen to dislodge from their respective antibodies. k_(on) is computedas k_(off)/K_(D). Antibody k_(on) and k_(off) values are summarized anddisplayed in a logarithmic plot.

Illumina generated sequences are analyzed using a battery of proprietaryalgorithms developed with Mathematica 8. A modified Hamming code is usedto construct tags that correct for single base errors. A tag isidentified in each DNA sequence and matched to the physical locations onthe NimbleGen chip. The NimbleGen locations are subsequently matched toscanning coordinates on the antibody capture slide.

The DNA sequences of the paired heavy and light chain of the murine IgGvariable domains mapped to those coordinates on the antibody captureslide identified by fluorescently labeled rhTNF-α will allowreconstruction of an antibody that binds to TNF-α. These identifiedmurine IgG variable domains are referred to as TNF-α binder candidates.The DNA sequences containing the human IL-6 gene with the tag identicalto that of a TNF-α binder candidate are collated; the random code ineach of these human IL-6 gene-containing sequences is compared amongeach other to assess the number of independent cDNA molecules todetermine the ACN of the human IL-6 gene mRNA associated with amicrowell that identified one of the TNF-α binder candidate. Similarly,the DNA sequences containing the human GAPDH gene with the tag identicalto that of the TNF-α binder candidates are also collated; the randomcode in each of these human GAPDH-containing sequences is compared amongeach other to assess the number of independent cDNA molecules todetermine the ACN of the human GAPDH gene mRNA. Having available the ACNof human IL-6 gene mRNA and the ACN of human GAPDH gene mRNA forassociated with a TNF-α binder candidate, the NCN of the human IL-6 genemRNA associated with a TNF-α binder candidate can be computed and theactivity of such a TNF-α binder candidate functions as a modulator canbe assessed.

The preceding merely illustrates the principles of the invention. Itshould be appreciated that those skilled in the art will be able todevise various arrangements which, although not explicitly described orshown herein, embody the principles of the invention and are includedwithin its spirit and scope. Furthermore, all examples and conditionallanguage recited herein are principally intended to aid the reader inunderstanding the principles of the invention and the conceptscontributed by the inventors to furthering the art, and are to beconstrued as being without limitation to such specifically recitedexamples and conditions. Moreover, all statements herein recitingprinciples, aspects, and embodiments of the invention as well asspecific examples thereof, are intended to encompass both structural andfunctional equivalents thereof. Additionally, it is intended that suchequivalents include both currently known equivalents and equivalentsdeveloped in the future, i.e., any elements developed that perform thesame function, regardless of structure. The scope of the presentinvention, therefore, is not intended to be limited to the exemplaryembodiments shown and described herein. Rather, the scope and spirit ofpresent invention is embodied by the appended claims.

1. A method of measuring a response of a reporter cell to a binding of aligand to a receptor comprising the steps of: placing a plurality ofligands into microwells; placing one or more reporter cells of one ormore types into the microwells occupied by the ligands; allowing theligands to interact with a receptor of the reporter cells; lysing thereporter cells in the microwells; capturing mRNA from one or more ofreporter genes of the reporter cells and mRNA from one or morehousekeeping genes of the reporter cells from the microwells with anoligonucleotide capture support containing mRNA capture oligos where themRNA capture oligos comprise a DNA tag unique to each microwell and arandom code; converting captured mRNA into cDNA incorporating the DNAtag and random code; sequencing the tagged cDNA by next generationsequencing (NGS); examining the sequenced cDNA from the one or morereporter genes of the reporter cells; using the DNA tags and randomcodes to compute an Absolute Copy Number (ACN) of the reporter genes andan ACN of housekeeping genes for the reporter cells in each well; usinga ratio of the ACN of the reporter genes to the ACN of the housekeepinggenes to compute a Normalized Copy Number (NCN) of the reporter genesfor the reporter cells in each well; examining the mRNA sequencesencoding the reporter genes from the reporting cells in each well; andfor each well, associating the mRNA sequences encoding the reportergenes from the reporter cells with the NCN of the mRNA from the reportergenes.
 2. The method of claim 1 wherein the number of nucleotides in therandom code is more than 4 nucleotides.
 3. The method of claim 1 whereinthe receptor is a membrane bound protein permanently bound to a lipidbilayer of the reporter cell, a peripheral membrane protein temporarilyassociated with the lipid bilayer or integral membrane protein, or alipid-anchored protein bound to the lipid bilayer through lipidatedamino acid residues.
 4. The method of claim 1, wherein the volume of themicrowells is between 10 and 1000 picoliters.
 5. The method of claim 1,wherein the number of nucleotides in the DNA tag is more than 6nucleotides.
 6. The method of claim 1, wherein the number of reportercells deposited into said microwells is between 1 and 500 cells permicrowell.